WO2005067506A2 - Procede pour controler et produire un vecteur adenoviral n'exprimant plus de genes viraux avec un rendement eleve - Google Patents

Procede pour controler et produire un vecteur adenoviral n'exprimant plus de genes viraux avec un rendement eleve Download PDF

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WO2005067506A2
WO2005067506A2 PCT/US2004/043877 US2004043877W WO2005067506A2 WO 2005067506 A2 WO2005067506 A2 WO 2005067506A2 US 2004043877 W US2004043877 W US 2004043877W WO 2005067506 A2 WO2005067506 A2 WO 2005067506A2
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helper
recombinase
adenovirus
site
dependent
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PCT/US2004/043877
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WO2005067506A3 (fr
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John S. Yu
Gentao Liu
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Cedars-Sinai Medical Center
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Publication of WO2005067506A3 publication Critical patent/WO2005067506A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT

Definitions

  • the invention relates to the production of gutless adenovirus vectors useful in gene therapy applications.
  • adenovirus vectors have become important tools in gene therapy for transfer of heterologous, therapeutic genes to diseased cells or tissues.
  • Adenovirus is an attractive candidate for gene therapy vectors because it can be produced in high titer stocks; adenovirus can infect resting and nondividing cells including dendritic cells and neurons; and the linear, double- stranded DNA of the adenoviral genome can be manipulated to accommodate foreign genes that range in size.
  • adenovirus does not require host cell proliferation to express either transgene-encoded or its own proteins; adenovirus has little to no toxicity with humans; and adenoviral vectors do not effect a cell's normal function because it does not insert into the chromosome of a cell.
  • Human adenovirus has been used in humans as an in vivo gene delivery vehicle and it is a promising tool for treatment of genetic diseases and cancer.
  • One drawback to early ' adenovirus-mediated gene therapy was that first generation adenovirus vectors were not suitable for long term stable transgene expression. Low-level expression of viral genes is, to a large extent, responsible for direct vector toxicity, inflammation in the transduced tissue, and a strong cellular immune response against the virus.
  • adenovirus vectors deleted of all viral coding sequences also known as high capacity "gutless” adenovirus or helper-dependent adenovirus
  • High capacity "gutless” adenoviral vectors are designed to retain only the elements required in cis for replication and packaging including inverted terminal repeats (ITR) flanking the genome for DNA replication, an adenoviral packaging signal to effect insertion of the completed viral genome into a completed viral capsid, and a heterologous transgene.
  • ITR inverted terminal repeats
  • adenoviral packaging signal to effect insertion of the completed viral genome into a completed viral capsid
  • a heterologous transgene a heterologous transgene. Growth of gutless adenovirus then depends on the assistance of a helper virus, which provides all necessary adenoviral proteins in trans.
  • the first efficient and currently widely used means for generating helper- dependent "gutless" adenovirus vectors is the Cre/loxP system. That system employed a recombinase, such as Cre, expressed by a cell into which a helper virus, comprising loxP sites flanking the adenovirus packaging signal, was introduced. In this system, Cre-expressing 293 cells are coinfected with helper-dependent vector and a helper virus bearing a packaging signal flanked by loxP sites. By virtue of the recombinase expressed by the host cell, the helper adenovirus packaging signal is excised, thereby restricting the packaging of the helper virus.
  • Cre/loxP system employed a recombinase, such as Cre, expressed by a cell into which a helper virus, comprising loxP sites flanking the adenovirus packaging signal, was introduced.
  • Cre-expressing 293 cells are coinfected with helper-dependent vector and a helper virus bearing a packaging signal flanked
  • the risk of rearrangements is even greater because of (i) the co-replication of two different genomes in a single infected cell resulting in extremely high concentrations of donor and target sequences within replication centers, (ii) partial sequence overlap between these genomes, and (iii) the absence of selection pressure to maintain the structure of the helper-dependent vector. While not wishing to be bound by any particular theory, it is believed that there is not enough Cre recombinase within the coinfected 293 cells during the helper-dependent vector generation to efficiently render all the helper virus genome unpackageable because Cre expression by the stable-transfected 293 cells can be shut off or markedly diminished due to virus infection.
  • helper-dependent adenoviral vector generation Another problem associated with helper-dependent adenoviral vector generation is that there is currently no way to monitor the generating process for optimization of each passaging of helper-dependent vector generation. This is partially due to the fact that helper-dependent vectors can not be quantified like the first generation adenovirus vectors. For the co-infection process, there is usually a multiplicity of infection (MOI) of 3 to 5 for helper virus but no MOI is available for the helper-dependent vector.
  • MOI multiplicity of infection
  • an adenovirus vector system for producing packaged adenovirus virions that express foreign nucleic acid sequences.
  • the helper virus includes inverted terminal repeats flanked by recombinase specific excision signals. Furthermore, the helper virus of the vector system expresses a site-specific recombinase gene for excising the helper virus packaging signal.
  • a helper-dependent adenovirus vector is also provided with up to 35kb of the adenovirus genome removed and up to a 35kb foreign nucleic acid inserted in the genome.
  • a method for producing a self-limiting helper virus wherein the helper virus has inverted terminal repeats flanked by recombinase specific excision signals. Furthermore, the helper virus of the vector system expresses a site-specific recombinase gene for excising the helper virus packaging signal.
  • a method for producing a packaged helper-dependent adenovirus vector is provided. The method includes coinfecting a cell with helper virus and helper-dependent adenovirus vectors. The helper virus includes inverted terminal repeats flanked by recombinase specific excision signals.
  • helper virus of the vector system expresses a site-specific recombinase gene for excising the helper virus packaging signal.
  • a helper-dependent adenovirus vector is also provided with up to 35kb of the adenovirus genome removed and up to a 35kb foreign nucleic acid inserted in the genome.
  • a method for monitoring the production of helper-dependent adenovirus vector is provided wherein the presence of helper virus genome in the helper-dependent adenovirus vector is assessed between serial passaging using electrophoresis and fluorescent polymerase chain reaction analysis.
  • Helper virus with a packaging signal flanked by loxP sites in the E1 region and a Cre cassette in the E3 region are infected into a 293 cell medium for vector propagation.
  • Dox is added to the 293 medium to modulate the expression of Cre by the helper virus upon serial passage and replication of the helper-dependent adenovirus vector.
  • Dox is added to the medium to turn off the expression of Cre recombinase, resulting in a packageable helper virus genome.
  • FIGURE 2 depicts a step-wise helper virus and helper-dependent vector packaging mechanism in first generation infection and transfection. Helper virus and helper-dependent vector are added to 293 medium for vector serial passaging.
  • FIGURE 3 depicts a step-wise helper virus and helper-dependent vector packaging mechanism in serial passage generation coinfection. Helper-dependent vector is added to 293 medium for vector serial passaging after already undergone first generation passage. The helper-dependent vector is efficiently packaged with the desired transgene through the assistance of the helper virus.
  • FIGURE 4 depicts a step-wise helper virus and helper-dependent vector stability analysis. Following coinfection of helper virus and helper-dependent vector in 293 medium, virus DNA is extracted for PCR analysis with fluorescent labeled forward primer.
  • Analysis of the stability of the gutless adenovirus generating system is determined by electrophoresis of the PCR products and quantifying fluorescence intensity of the bands that represent packaging signal-bearing and -excised helper virus genomes, and replicated HD vector genome respectively.
  • the present invention is based on a novel system to overcome helper virus overgrowth in the production of "gutless" adenovirus vectors for gene therapy. While not wishing to be bound by any theory, it is believed that helper-dependent adenovirus production and helper virus overgrowth are limited by adenovirus- mediated recombinase shutoff.
  • the present invention addresses this problem by expressing recombinase within the helper virus genome itself, along with a packaging signal flanked by excision sites that are the target sequences of the recombinase. Thus, the helper virus renders itself unpackageable by the expression of recombinase from its own genome.
  • the present invention further addresses this problem by employing a novel means of monitoring the production of helper virus and helper-dependent adenovirus vector through the use of electrophoresis combined with fluorescent PCR.
  • Any publications referenced herein are hereby incorporated by reference in this application in order to more fully describe the state of the art to which the present invention pertains. It will be understood that all technical and scientific terms used to describe the present invention herein, unless otherwise defined, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. The techniques employed herein are also those that are known to one of ordinary skill in the art, unless stated otherwise. Recombinant adenoviral vectors currently in use typically have deletions in the E1 , E2 or E4 regions.
  • the high-capacity gutless adenoviral vector generating method described herein uses vectors that have a self-limiting recombinase expressed by the viral genome, such that, the helper adenovirus provides all of the functions necessary for viral replication and packaging, but is itself unable to be packaged and the helper-dependent adenovirus vector is replicated and packaged into infectious virions.
  • Previous helper virus systems suffer from residual contamination when rendered unpackageable by the Cre-mediated excision of a loxP flanked packaging signal ( ⁇ ).
  • helper-dependent adenoviral vector requires three regions of the viral DNA in cis, including the left and right terminal repeats (ITR) and the packaging signals. All other viral DNA is required for in trans replication of the virus. Helper-dependent adenovirus, in this fashion, can have all viral DNA removed other than the required in cis regions. Helper virus provides all the necessary viral products for in trans production and replication.
  • FLP recombinase which recognizes the sequences known as FRT
  • Cre recombinase which recognizes the sequence known as loxP
  • FRT FLP recombinase
  • loxP Cre recombinase
  • Cre or loxP such mention should be read to include any other site- specific recombination system now known or henceforth discovered, when applied to the specific techniques described herein.
  • the terms used herein are not intended to be limiting to the invention.
  • the term "gene” includes DNAs, cDNAs, RNAs, or other polynucleotides that encode gene products.
  • Form gene denotes a gene that has been obtained from an organism or cell type other than the organism or cell type in which it is expressed; it also refers to a gene from the same organism that has been translocated from its normal situs in the genome.
  • nucleic acid RNA
  • DNA DNA
  • nucleic acid analogues and derivatives can be made and will hybridize to one another and to DNA and RNA, and the use of such analogues and derivatives is also within the scope of the present invention.
  • "Expression" of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acids(s) in cloning systems and in any other context.
  • Recombinase encompasses enzymes that induce, mediate or facilitate recombination, and other nucleic acid modifying enzymes that cause, mediate or facilitate the rearrangement of a nucleic acid sequence, or the excision or insertion of a first nucleic acid sequence form or into a second nucleic acid sequence.
  • the "target site" of a recombinase is the nucleic acid sequence or region that is recognized (excised, cut or induced to recombine) by the recombinase.
  • gene product refers primarily to proteins and polypeptides encoded by a nucleic acid, but further encompasses nucleic acids encoded by other nucleic acids (e.g., non-coding and regulatory RNAs such as transfer RNA (tRNA) and small ribonucleoprotein particles (sNRPs)).
  • RNAs such as transfer RNA (tRNA) and small ribonucleoprotein particles (sNRPs)
  • regulation of expression refers to events or molecules that increase or decrease the synthesis, degradation, availability or activity of a given gene product.
  • the helper virus and helper- dependent adenovirus are included in separate plasmids for transfecting cells.
  • Mammalian cell lines which express recombinases are suitable for use with the present invention.
  • a mammalian cell line which expresses the Cre recombinase may be particularly suitable for use in connection with various embodiments of the present invention.
  • a cell line expressing Cre recombinase can be coinfected with the helper virus and helper-dependent vector.
  • One example of such a cell line is the human embryonic kidney (HEK) 293 cell line. Upon infection of 293 cells, the packaging signal of adenovirus is efficiently excised from approximately 90% of the helper virus DNA.
  • HEK human embryonic kidney
  • helper virus and helper-dependent adenovirus may be transfected into GTM-3 medium (available from Sigma; St. Louis, MO), Adenovirus Expression Medium (AEM) (available from Invitrogen; Carlsbad, CA), Ex-cell VPRO Medium (available from JRH Biosciences; Lenexa, KS), and DMEM (available from Sigma).
  • GTM-3 medium available from Sigma; St. Louis, MO
  • AEM Adenovirus Expression Medium
  • Ex-cell VPRO Medium available from JRH Biosciences; Lenexa, KS
  • DMEM available from Sigma
  • the present invention is not limited to the use of the cell types and cell lines used herein. Different tissue sources, for example, breast, epithelium, colon, or lymphocytes and different species, for example, human or mouse, are also suitable for use with the present invention.
  • the recombinase of the present invention may be provided in trans, via a third plasmid, or in cis, by inclusion of a recombinase expression cassette in one of the introduced plasmids.
  • the invention also includes a transgene regulatable system, Tet-off, in the helper virus genome.
  • Tet-off expression systems are binary transgenic systems that employ an inducible transcriptional activator to regulate the expression of a target transgene. Expression of the transcriptional activator can be regulated by exposure to varying concentrations of tetracycline or a derivative such as doxycycline, both of which inactivate transcription.
  • transgene regulatable system allows the helper virus to grow for co-infection.
  • the Tet-off system is used to turn off Cre expression during propagation of the helper virus, and may thereafter be used to turn on Cre expression during helper-dependent vector propagation.
  • the helper virus is thereby inhibited from being packaged and contaminating the helper-dependent vector during the vector propagating process.
  • Further embodiments of the present invention include inverting the packaging signal orientation of the helper virus genome to help avoid recombination between the helper virus and helper-dependent vector genome, as recombination may result in the helper virus genome losing one of the loxP sites and thereby becoming resistant to Cre.
  • the present invention further includes a method for constructing viruses, plasmids or both which contain viral DNA based on the Cre/loxP system first reported in U.S. Patent No. 6,080,569, which is a continuation-in-part of U.S. Pat. No. 5,919,676, which is a continuation-in-part of U.S. patent application Ser. No. 08/250,885, filed on May 31 , 1994, which is a continuation-in-part of abandoned U.S. patent application Ser. No. 08/080,727, filed June 24, 1993. All of the aforementioned applications are hereby incorporated by reference in their entirety as if fully set forth.
  • helper viruses that express Cre using a transgene regulatable system, Tet-off or Lac-off
  • viral DNA may also contain loxP sites positioned such that site-specific recombination between loxP sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in co-transfected host cells.
  • suitably engineered plasmids can be readily recombined to produce infectious virus at high efficiency in co- transfected 293 cells, without, at the same time, producing a contaminating parental adenovirus, with the attendant problems for removal thereof.
  • Adenoviral vectors based on this model are well known in the art, for example, U.S. Patent No. 6,566,128 describes various adenoviral models, and is hereby incorporated by reference in its entirety.
  • the assembly and production of the gutless adenovirus of the present invention may be completed in three stages.
  • the first step involves helper virus propagation in, for example, 293-derived cell lines that stably express Cre recombinase as depicted in Figure 1. This may be accomplished by the introduction of a plasmid containing helper virus into HEK 293 cell medium with doxycycline added to switch off Cre expression by the helper genome.
  • helper virus in construction of the helper virus, the packaging signal ( ⁇ ) orientation is inverted to prevent recombination between the helper virus and the helper-dependent adenovirus.
  • recombinant helper-dependent adenovirus vector may be packaged into infectious adenovirus by transfecting HEK 293 cells as depicted in Figure 2.
  • the packaging signal ( ⁇ ) of the helper virus genome is flanked by loxP sites that are the target of Cre excision. Cre expression is switched on by eliminating doxcycline from the 293 cell line medium, thereby making helper virus unpackageable during co-transfection.
  • Co-transfection is implemented to accommodate DNA inserts in adenoviral vector because extensive portions of the early regions 1 (E1) and 3 (E3) of wild-type adenovirus are deleted from the adenovirus genome. Eliminating the E1 elements generally requires an early passage HEK 293 cell line to propagate the recombinant adenoviruses (Graham et al., 1977; Aiello et al., 1979), because 293 cells stably express the adenovirus E1 genes that are essential for replication and transcription of adenovirus DNA.
  • the recombinant adenovirus may be harvested by lysing transfected cells, followed by further passages of infecting 293 cells with helper virus and recombinant helper-dependent adenovirus vector as depicted in Figure 3.
  • the genomes of the helper-dependent vectors need contain only those sequences required in cis for viral replication: the Ad ITRs and packaging signal, comprising approximately 500 bp of Ad DNA.
  • other viral sequences may be present, as well as stuffer sequences and other DNA segments encoding foreign genes and regulatory elements such as promoters, enhancers and polyadenylation signals.
  • Helper-dependent titer can be increased by serial passage with helper virus in 293 medium.
  • Helper virus is grown in the 293 cell line, which will complement the recombinase gene incorporated in the helper virus genome further aiding in the excising of the viral packaging signal, thereby rendering the virus unpackageable. Further, the packaging signal of the helper virus is inverted to prevent recombination with the helper-dependent adenoviral genome. In this manner, two separate mechanisms, Cre-mediated excision of the viral packaging signal and the inverted packaging signal function to ensure complete excision of the helper virus packaging signal and elimination of all helper virus from vector preparations.
  • a method of monitoring the production of adenovirus vectors is included.
  • the method of the invention is based on a fluorescent polymerase chain reaction ( *f PCR) method that can quantify both the packaging signal-excised and signal-bearing helper virus genome together with replicated helper-dependent vector genomes during the co-infection passaging process. Based on the fact that the packaging signal-excised and signal-bearing helper virus genome are identical except for the approximately 180 base pair difference of the packaging signal, it is not possible to quantify both genomes by real-time PCR.
  • kits comprising helper viruses that express Cre using a transgene regulatable system, Tet-off, helper-dependent virus, 293 cells and instructions for their use.
  • the exact nature of the components configured in the inventive kit depends on its intended purpose and on the particular methodology that is employed.
  • some embodiments of the kit are configured for the purpose of treating a genetic disorder in a subject.
  • the kit is configured particularly for the purpose of generating gutless adenovirus that may be used in the treatment of human subjects.
  • kits Instructions for use may be included with the kit.
  • “Instructions for use” typically include a tangible expression describing the steps for preparing gutless adenovirus and/or for using the same in a therapeutic system.
  • the kit also contains other useful components, such as diluents, buffers, pharmaceutically acceptable carriers, specimen containers, syringes, stents, catheters, pipetting or measuring tools, paraphernalia for concentrating, sedimenting, or fractionating samples, or antibodies and/or primers and/or probes for controls.
  • the materials or components assembled in the kit can be provided to the practitioner stored in any convenient and suitable ways that preserve their operability and utility.
  • the components can be in dissolved, dehydrated, or lyophilized form; they can be provided at room, refrigerated, or frozen temperatures.
  • the components are typically contained in suitable packaging material(s).
  • packaging material refers to one or more physical structures used to house the contents of the kit.
  • the packaging material is constructed by well known methods, preferably to provide a sterile, contaminant-free environment.
  • the packaging materials employed in the kit are those customarily utilized in the field.
  • the term "package” refers to a suitable solid matrix or material such as glass, plastic, paper, foil, and the like, capable of holding the individual kit components.
  • a package can be a glass vial used to contain suitable quantities of a composition containing the 293 cells or a medium.
  • the packaging material generally has an external label which indicates the contents and/or purpose of the kit and/or its components. Therefore, it is an object of the present invention to provide an efficient, reliable and simple method for isolation of viral vectors in which rescue of viruses is enhanced by provision of Cre expression on helper virus. It is a further object of the invention to provide a simple and useful system by which adenovirus cloning vectors may be developed. It is a further object of the invention to provide a kit for efficient production of adenoviral vectors for vaccine and gene-therapeutic applications which relies on Cre expression on helper virus genome.
  • a method for treating a disease condition in mammals may include providing an adenoviral helper-dependent vector; providing a helper virus; providing a transgene useful in connection with a gene therapy; and implementing a gene therapy regimen with the aforementioned vector and transgene in a manner to treat the particular condition.
  • the helper-dependent vector and helper virus may have characteristics similar to the compositions described above in accordance with alternate embodiments of the present invention.
  • the methods of the present invention are not limited to the treatment of any particular disease condition.
  • the methods of the present invention may find application in the treatment of any disease condition in which treatment with an adenoviral vector and transgene may cause a beneficial result for a patient is thus included within the scope of the present invention.
  • a composition including both an adenoviral vector and an additional immunoregulatory molecule of rather than administering these compounds separately in a combination therapy. This is considered to be within the ambit of the present invention.
  • a composition might have superior characteristics as far as clinical efficacy, solubility, absorption, stability, toxicity and/or patient acceptability are concerned.
  • a therapeutically effective dose refers to that amount of active ingredient which increases or decreases the effects of a disease condition relative to that which occurs in the absence of the therapeutically effective dose.
  • Therapeutic efficacy and toxicity e.g., ED 50 (the dose therapeutically effective in 50% of the population) and LD 50 (the dose lethal to 50% of the population), can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED5 0 .
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use, which can be readily tended to by one of ordinary skill in the art without undue experimentation.
  • the dosage contained in such compositions may be selected so as to be within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

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Abstract

Cette invention se rapporte à un adénovirus de cellules assistantes et à des vecteurs d'adénovirus dépendant de cellules assistantes de rendement élevé pour l'encapsidation et l'expression d'acides nucléiques étrangers. Les vecteurs faisant l'objet de cette invention sont basés sur un virus de cellules assistantes à autolimitation qui est conçu pour éliminer la contamination de titrages. Cette invention concerne en outre un procédé pour contrôler la production de vecteurs d'adénovirus dépendant de cellules assistantes pour éliminer la contamination de virus de cellules assistantes.
PCT/US2004/043877 2004-01-06 2004-12-29 Procede pour controler et produire un vecteur adenoviral n'exprimant plus de genes viraux avec un rendement eleve WO2005067506A2 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001591A (en) * 1993-12-09 1999-12-14 University Of Saskatchewan Recombinant bovine adenoviruses
US6541245B1 (en) * 1999-09-23 2003-04-01 Genzyme Corporation Adenoviral helper vectors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6001591A (en) * 1993-12-09 1999-12-14 University Of Saskatchewan Recombinant bovine adenoviruses
US6541245B1 (en) * 1999-09-23 2003-04-01 Genzyme Corporation Adenoviral helper vectors

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
PARKS ET AL: 'A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal.' PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES. vol. 93, November 1996, pages 13565 - 13570, XP002201914 *
SANDIG ET AL: 'Optimization of the helper-dependent adenovirus system for production and potency in vivo.' PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES. vol. 97, no. 3, February 2000, pages 1002 - 1007, XP002927564 *

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